Protein radical formation resulting from eosinophil peroxidase-catalyzed oxidation of sulfite.
Journal - The Journal of biological chemistry
Eosinophil peroxidase (EPO) is an abundant heme protein in eosinophils that catalyzes the formation of cytotoxic oxidants implicated in asthma, allergic inflammatory disorders, and cancer. It is known that some proteins with peroxidase activity (horseradish peroxidase and prostaglandin hydroperoxidase) can catalyze oxidation of (bi)sulfite (hydrated sulfur dioxide), leading to the formation of sulfur trioxide anion radical (.SO3-). This free radical further reacts with oxygen to form peroxymonosulfate anion radical (-O3SOO.) and the very reactive sulfate anion radical (SO4.-), which is nearly as strong an oxidant as the hydroxyl radical. However, the ability of EPO to generate reactive sulfur radicals has not yet been reported. Here we demonstrate that eosinophil peroxidase/H2O2 is able to oxidize (bi)sulfite, ultimately forming the sulfate anion radical (SO4.-), and that these reactive intermediates can oxidize target proteins to protein radicals, thereby initiating protein oxidation. We used immuno-spin trapping and confocal microscopy to study protein oxidation by EPO/H2O2 in the presence of (bi)sulfite in a pure enzymatic system and in human promyelocytic leukemia HL-60 clone 15 cells, maturated to eosinophils. Polyclonal antiserum raised against the spin trap 5,5-dimethyl-1-pyrroline N-oxide (DMPO) detected the presence of DMPO covalently attached to the proteins resulting from the DMPO trapping of protein free radicals. We found that sulfite oxidation mediated by EPO/H2O2 induced the formation of radical-derived DMPO spin-trapped human serum albumin (HSA) and, to a lesser extent, of DMPO-EPO. These studies suggest that EPO-dependent oxidative damage may play a role in tissue injury in (bi)sulfite-exacerbated eosinophilic inflammatory disorders.